Adult teleost fish and urodele amphibians can regenerate entire amputated appendages. By contrast, regenerative healing of adult mammalian limbs is limited to the very tips of digits. One of the key challenges in developmental biology is to understand how and why tissue regeneration occurs. The hallmark of limb or fin regeneration is formation of a blastema, a mesenchymal structure that contains progenitor cells for new skeletal elements. As regeneration proceeds, blastemal cell proliferation and patterning are regulated such that lost tissues of correct size and shape are replaced, a phenomenon called positional memory. Skeletal osteoblasts in the blastema and, ultimately, the regenerated fin rays, derive from the proliferation of pre- existing osteoblasts, while an alternative source(s exists if the primary osteoblast source is disabled. Activating and inhibitory factors influence regenerative growth, and region-specific maintenance of patterning transcription factors in fin cells throughout life is a component of positional memory. Despite recent advances in our understanding of appendage regeneration, there remain key deficiencies in the field. First, there have been no published attempts to image and quantify the in vivo dynamics of cell proliferation during appendage regeneration, analyses that would illuminate how complex pattern is restored after injury. Second, a limited set of traditional developmental signaling factors has been assessed for function during regeneration. The overall goal of this proposal is to define mechanisms that regulate cell proliferation as size and pattern are restored to an amputated appendage. 1) We will apply new technologies to monitor in vivo cell cycle progression and growth indicators in transgenic zebrafish, to create a spatiotemporal map of cell proliferation during fin regeneration. 2) We will use new reagents to define whether and how signaling by the secreted factor Leptin influences zebrafish fin regeneration. 3) We will define mechanisms by which two genes implicated from a mutagenesis screen, the extracellular matrix component laminin beta 1a and the voltage-gated calcium channel calcium homeostasis modulator 1, influence fin regeneration. With these approaches, we will test the hypothesis that blastemal cell proliferation is controlled by a complex microenvironment of activating and inhibitory influences. This work will increase understanding of developmental regulation during vertebrate tissue regeneration, and provide important perspective for comprehending, and perhaps changing, the existing limitations in regenerative capacity of human tissues.